Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Induction of differentiation using the Ex.1 method. (A) Colony of human iris-derived iPS cells. (B-D) Cell colony in differentiation culture: (B) 6th day in STEP-1 medium, (C) 18th day in STEP-2 medium, and (D) 25th day in STEP-3 medium. Scale bar in (A-D), 100 µm. (E) Fold-increase in p75NTR mRNA expression level at different steps relative to STEP-0: * P<0.05. (F) Cell morphology on 35th day of differentiation in STEP-3 medium. (G) Immunostaining for αA-crystallin in the same area as indicated in (F). (H) Immunostaining for SOX2 in the same area as in (G). (I) Merged image of αA-crystallin and SOX2 immunostaining. Scale bar in (F-I), 100 µm. Ex, experiment; iPS cells, induced pluripotent stem cells.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Derivative Assay, Expressing, Immunostaining

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Changes in gene expression levels at each step of differentiation induction using Ex.4 the method. (A) SOX2 , (B) PAX6 , (C) p75NTR , (D) αA-crystallin , (E) type-IV collagen ; fold-changes are relative to gene expression level in STEP-0: * P<0.05, ** P<0.01, *** P<0.005. Ex, experiment.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Induction of differentiation using the Ex.4 method. (A) Gross observation of cell aggregate. Reduced opacity is observed in certain areas. Scale bar, 2 mm. (B) H&E-stained specimen of cell aggregate. Scale bar, 200 µm. (C) Enlarged view of H&E staining in Area 1 of the specimen in (B). Cells displaying epithelial-like morphology have infiltrated inside the cell aggregate (yellow arrowhead). (D-G and I-L) Immunostaining of cell aggregates for (D and I) SOX2, (E and J) p75NTR, (F and K) αA-crystallin, and (G and L) type IV collagen. Blue in immunostaining images: DAPI staining of nuclei. (H) Enlarged view of H&E staining of Area 2 in the specimen in (B), where the cells on the surface of the aggregate have transitioned from a multilayer to a monolayer (white arrowhead). Scale bar in (C-L), 50 µm. Ex, experiment; H&E, hematoxylin and eosin.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Staining, Immunostaining

Journal: Cell Stem Cell

Article Title: Selective advantage of mutant stem cells in human clonal hematopoiesis is associated with attenuated response to inflammation and aging

doi: 10.1016/j.stem.2024.05.010

Figure Lengend Snippet:

Article Snippet: APC anti-human CD271 (clone ME20.4-1.H4) , Miltenyi , Cat# 130-113-418; RRID: AB_2733363.

Techniques: Recombinant, Reverse Transcription, DNA Library Preparation, DNA Sequencing, RNA Sequencing, Purification, Sequencing, Software, Sterility

Journal: Journal of Clinical Medicine

Article Title: Moderate Aerobic Exercise Regulates Follicular Dysfunction by Initiating Brain-Derived Neurotrophic Factor (BDNF)-Mediated Anti-Apoptotic Signaling Pathways in Polycystic Ovary Syndrome

doi: 10.3390/jcm11195584

Figure Lengend Snippet: Sequences of primers designed for RT-qPCR.

Article Snippet: Sections were incubated overnight at 4 °C with antibodies against BDNF (1:100, Wanleibio), TrkB (1:100, Wanleibio), p75NTR (1:100, 55014-1-AP, Proteintech, Chicago, IL, USA), and Cleaved-Caspase-3 (1:100, 66470-2-Ig, Proteintech) at a 1:100 dilution.

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Moderate Aerobic Exercise Regulates Follicular Dysfunction by Initiating Brain-Derived Neurotrophic Factor (BDNF)-Mediated Anti-Apoptotic Signaling Pathways in Polycystic Ovary Syndrome

doi: 10.3390/jcm11195584

Figure Lengend Snippet: The expression of BDNF, TrkB, and p75NTR in ovarian follicles of PCOS rats after aerobic exercise. Rats received DHEA for the induction of polycystic ovarian syndrome, together with or without exercise treatment. ( A , B ) The expression levels of BDNF in ovarian follicles at different stages were analyzed by immunohistochemistry. ( C , D ) The expression levels of BDNF in ovarian follicles were analyzed by immunofluorescence. ( E , F ) The co-expression of TrkB and p75NTR in ovarian follicles at different stages was detected by immunofluorescence double labeling. n = 10 in each group. Data are shown as mean ± SEM. ** p < 0.05, vs. control group; ## p < 0.01, vs. PCOS group.

Article Snippet: Sections were incubated overnight at 4 °C with antibodies against BDNF (1:100, Wanleibio), TrkB (1:100, Wanleibio), p75NTR (1:100, 55014-1-AP, Proteintech, Chicago, IL, USA), and Cleaved-Caspase-3 (1:100, 66470-2-Ig, Proteintech) at a 1:100 dilution.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Labeling, Control

Journal: Journal of Clinical Medicine

Article Title: Moderate Aerobic Exercise Regulates Follicular Dysfunction by Initiating Brain-Derived Neurotrophic Factor (BDNF)-Mediated Anti-Apoptotic Signaling Pathways in Polycystic Ovary Syndrome

doi: 10.3390/jcm11195584

Figure Lengend Snippet: Aerobic exercise activated BDNF signaling in the ovary of DHEA-induced PCOS rats. Rats received DHEA for the induction of polycystic ovarian syndrome, together with or without exercise treatment. ( A ) mRNA expression of BDNF, TrkB, and p75NTR factors in ovarian tissue was analyzed by real-time PCR. ( B , C ) The expression of BDNF, TrkB, and p75NTR in ovarian tissue was assessed by Western blot assay. ( D , E ) The expression of PI3K, AKT, and p-AKT in ovarian tissue was assessed by Western blot assay. ( F – I ) The expression of p75NTR, NF-κB, p-NF-κB, JNK, and p-JNK in ovarian tissue was assessed by Western blot assay. n = 10 in each group. Data are shown as mean ± SEM. ** p < 0.05, vs. control group; ## p < 0.01, vs. PCOS group.

Article Snippet: Sections were incubated overnight at 4 °C with antibodies against BDNF (1:100, Wanleibio), TrkB (1:100, Wanleibio), p75NTR (1:100, 55014-1-AP, Proteintech, Chicago, IL, USA), and Cleaved-Caspase-3 (1:100, 66470-2-Ig, Proteintech) at a 1:100 dilution.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Brain Sciences

Article Title: NGFR Gene and Single Nucleotide Polymorphisms, rs2072446 and rs11466162, Playing Roles in Psychiatric Disorders

doi: 10.3390/brainsci12101372

Figure Lengend Snippet: NGFR SNPs analyzed in this study.

Article Snippet: We used a vector, pCMV6- Ngfr -cDNA, containing a full-length mice Ngfr cDNA clone purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: Variant Assay

Journal: Brain Sciences

Article Title: NGFR Gene and Single Nucleotide Polymorphisms, rs2072446 and rs11466162, Playing Roles in Psychiatric Disorders

doi: 10.3390/brainsci12101372

Figure Lengend Snippet: NGFR shRNA and control sequences.

Article Snippet: We used a vector, pCMV6- Ngfr -cDNA, containing a full-length mice Ngfr cDNA clone purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: shRNA, Control

Journal: Brain Sciences

Article Title: NGFR Gene and Single Nucleotide Polymorphisms, rs2072446 and rs11466162, Playing Roles in Psychiatric Disorders

doi: 10.3390/brainsci12101372

Figure Lengend Snippet: Allele and genotype distribution of NGFR gene polymorphisms between SCZ patients and healthy participants.

Article Snippet: We used a vector, pCMV6- Ngfr -cDNA, containing a full-length mice Ngfr cDNA clone purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: Control

Journal: Brain Sciences

Article Title: NGFR Gene and Single Nucleotide Polymorphisms, rs2072446 and rs11466162, Playing Roles in Psychiatric Disorders

doi: 10.3390/brainsci12101372

Figure Lengend Snippet: Allele and genotype distribution of NGFR gene polymorphisms between MDD patients and healthy participants.

Article Snippet: We used a vector, pCMV6- Ngfr -cDNA, containing a full-length mice Ngfr cDNA clone purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: Control

Journal: Brain Sciences

Article Title: NGFR Gene and Single Nucleotide Polymorphisms, rs2072446 and rs11466162, Playing Roles in Psychiatric Disorders

doi: 10.3390/brainsci12101372

Figure Lengend Snippet: GFP expressed by the transfected cells. The 293T cells were co-transfected with shRNA plasmid and pCAGIG- Ngfr -cDNA.

Article Snippet: We used a vector, pCMV6- Ngfr -cDNA, containing a full-length mice Ngfr cDNA clone purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: Transfection, shRNA, Plasmid Preparation

Journal: Brain Sciences

Article Title: NGFR Gene and Single Nucleotide Polymorphisms, rs2072446 and rs11466162, Playing Roles in Psychiatric Disorders

doi: 10.3390/brainsci12101372

Figure Lengend Snippet: Down-regulation of NGFR in 293T cells. ( A ) Immunoblotting of protein extracted from 293T cells co-transfected with shRNA plasmid and pCAGIG- Ngfr -cDNA. ( B ) One-way ANOVA of the protein levels after normalization to β-actin; data showed as means ± SEM, ** p < 0.01.

Article Snippet: We used a vector, pCMV6- Ngfr -cDNA, containing a full-length mice Ngfr cDNA clone purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: Western Blot, Transfection, shRNA, Plasmid Preparation

Journal: Brain Sciences

Article Title: NGFR Gene and Single Nucleotide Polymorphisms, rs2072446 and rs11466162, Playing Roles in Psychiatric Disorders

doi: 10.3390/brainsci12101372

Figure Lengend Snippet: Down-regulation of NGFR in mouse hippocampus. ( A ) Lenti- NGFR -sh2 or control virus was injected into the dentate gyrus of adult mice. Two weeks after injection, extracts from hippocampus were immunoblotted. ( B ) One-way ANOVA of the protein levels after normalization to β-actin, data showed as means ± SEM, * p < 0.05.

Article Snippet: We used a vector, pCMV6- Ngfr -cDNA, containing a full-length mice Ngfr cDNA clone purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: Control, Virus, Injection

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Steady-state distribution and delivery to the plasma membrane of PLAP and NTR-PLAP in transfected FRT cells. (A) Schemes of PLAP and NTR-PLAP (a fusion protein containing the ectodomain of p75NTR and the C-terminal GPI attachment signal of PLAP). (B) FRT cells stably expressing PLAP or NTR-PLAP were grown to confluence and pulsed overnight with [35S]met–cys or [35S]cys, respectively. After surface biotinylation from the apical (Ap) and basolateral (BL) domains, cells were lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (C) FRT cells were labeled for 20 min with [35S]met–cys or [35S]cys. At different times of the chase, surface proteins were biotinylated from the apical (Ap) or the basolateral (BL) side. Cells were then lysed and immunoprecipitated with antibodies against PLAP or NTR and with streptavidin beads. Samples were run on 10% SDS-PAGE and detected by fluorography. PLAP and NTR-PLAP are directly sorted to the apical surface in FRT cells, whereas a small percentage of the two proteins is delivered with similar kinetics to the basolateral domain.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Transfection, Stable Transfection, Expressing, Immunoprecipitation, Labeling, SDS Page

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Pulse-chase analysis and purification on sucrose gradients of transmembrane forms of PLAP and p75NTR in transfected FRT cells. (A) Schemes of p75NTR and PLAP-PS321 (a fusion protein containing the ectodomain of PLAP and the transmembrane and the cytoplasmic domain of the PS321 NTR mutant). (B) Pulse-chase and TX-100 extraction of p75NTR and PLAP-PS321. FRT cells expressing p75NTR and PLAP-PS321 were labeled with [35S]cys or [35S]met–cys, respectively, and subsequently treated as described in Figure ​Figure2A.2A. (C) Purification of p75NTR- and PLAP-PS321–enriched fractions on sucrose density gradients. Labeled proteins of FRT cells expressing p75NTR and PLAP-PS321 were solubilized in TX-100 and purified as described in Figure ​Figure2B.2B. Both transmembrane proteins are soluble in TX-100 and do not float on sucrose density gradients.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Pulse Chase, Purification, Transfection, Mutagenesis, Expressing, Labeling

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Surface expression of GPI-anchored, secretory, and transmembrane proteins after FB1 treatment. (A) FRT cells stably expressing PLAP, NTR-PLAP, or gD1-DAF were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After 30 min of labeling with [35S]met–cys or [35S]cys and a 2-h chase, cells were surface biotinylated from the apical (Ap) or the basolateral (BL) domain, lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (B) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. Apical PLAP and NTR-PLAP, but not basolateral gD1-DAF, became unpolarized after FB1 treatment. (C) FRT cells expressing PLAP-sec, NTR-sec, p75NTR, or PLAP-PS321 were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After labeling with [35S]met–cys or [35S]cys for 20 min and a chase of 2 h, apical and basolateral media were collected separately and cells were surface biotinylated from apical or basolateral domains. Polarized secretion of the secretory forms was revealed by immunoprecipitation in the apical and basolateral medium (top two panels), whereas biotinylated transmembrane proteins were revealed by double immunoprecipitation with specific antibody and streptavidin beads (bottom two panels). (D) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. FB1 does not alter the sorting of transmembrane and secretory proteins.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Expressing, Stable Transfection, Labeling, Immunoprecipitation

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Surface expression of GPI-anchored and transmembrane proteins after cholesterol depletion. (A) FRT cells expressing PLAP, NTR-PLAP, gD1-DAF, p75NTR, or PLAP-PS321 were grown on filters and treated with mevinolin (Mev.; 10 μM) for 48 h and M-β-CD (10 mM) for 60 min. Cells were then pulse labeled for 30 min, chased for 2 h, and biotinylated as usual. (B) Amounts of labeled proteins were quantified in three independent experiments and are shown as means ± SD. Hatched bars represent apical (Ap) proteins, and black bars represent basolateral (BL) proteins. Removal of cholesterol does not alter the surface expression of apical and basolateral GPI-anchored and transmembrane proteins.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Expressing, Labeling